Glucosyltransferase mutant, construction method thereof, construction body, mutant expression strain, application and preparation method of rebaudioside M

Abstract

The invention belongs to the technical field of molecular biology, and discloses a glucosyltransferase mutant, a construction method of the glucosyltransferase mutant, a construction body, a mutant expression strain, application and a preparation method of rebaudioside M. Compared with original glucosyltransferase (comprising an amino acid fragment with the sequence shown as SEQ ID NO: 1), the glucosyltransferase mutant provided by the invention comprises one or more of L79T mutation, S195 V mutation and Y387V mutation. The glucosyltransferase mutant has excellent enzyme activity and can be cooperated with existing sucrose synthase to realize a multi-cascade enzyme catalytic reaction, and sucrose, rebaudioside D and uridine diphosphate are used as raw materials to realize high-efficiency and low-cost industrial production of rebaudioside M.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular biology, and in particular relates to a glucosyltransferase mutant and a construction method, a construct, a mutant expression strain, and an application thereof, and a preparation method of rebaudioside M. Background Art

[0002] Stevia is a herbaceous plant of the Asteraceae family originating from South America. The steviol glycosides it contains have the characteristics of high sweetness, extremely low calories and good stability, and are known as the "third generation healthy sugar source" and "the best natural sweetener". Stevioside glycosides are mainly found in stevia leaves, with the highest content being stevioside (accounting for 5-10% of the dry weight of the leaves), rebaudioside A (accounting for 2-4% of the dry weight of the leaves) and rebaudioside C (accounting for 1-2% of the dry weight of the leaves), while the remaining more than 50 glycosides such as rebaudioside D, rebaudioside M, rebaudioside E, etc. have extremely low content.

[0003] Stevioside is about 200 to 350 times sweeter than sucrose, and has only 200 to 300 times the calories of sucrose. It is a high-quality sweetener substitute and has been used in chocolate, yogurt, jam, and beverages. Studies have shown that stevioside is effective in the auxiliary treatment of diabetes, hypertension, colitis, cirrhosis, etc., so stevioside has a broad application space in the fields of food, beverages and medicine.

[0004] Steviosides have a common steviol unit. By adding different numbers and types of glycosyl groups at the C-13 and C-19 positions of the steviol unit, a variety of steviol glycosides with different tastes and physical and chemical properties are formed. Since stevioside and rebaudioside A have a bitter aftertaste, rebaudioside D and rebaudioside M are considered to be more ideal sweeteners. In terms of sweetness and taste, rebaudioside M is better than rebaudioside D and rebaudioside A.

[0005] Since the content of rebaudioside M in plants is extremely low, it cannot be extracted by plant extraction methods, and the chemical synthesis method is complex, energy-intensive and harmful to the environment, which is also not conducive to the large-scale production of rebaudioside M. Glucosyltransferase is an enzyme that only transfers glucose in an enzyme reaction. The mechanism of action of the enzyme is to catalyze the transfer of the glucose residue of the glycosyl donor to the glycosyl acceptor molecule, thereby regulating the activity of the acceptor molecule. Glucosyltransferase can be applied to the preparation of rebaudioside M to achieve its biocatalytic synthesis. The production of rebaudioside M by biocatalytic synthesis has the characteristics of large production scale, short time and environmental friendliness. However, the glucosyltransferases used in the current biocatalytic synthesis method are mostly wild enzymes derived from plant cells, which often have the disadvantage of low enzyme activity, resulting in high cost for its application in industrial large-scale production of rebaudioside M.

[0006] Therefore, it is of great significance to modify the wild glucosyltransferase derived from plant cells and effectively improve the enzyme activity, which is conducive to the industrial production of rebaudioside M. Summary of the invention

[0007] The first object of the present invention is to provide a glucosyltransferase mutant. The glucosyltransferase mutant has excellent enzyme activity and can cooperate with existing sucrose synthase to use sucrose, rebaudioside D and uridine diphosphate as raw materials to achieve high-efficiency and low-cost industrial production of rebaudioside M.

[0008] The second object of the present invention is to provide a method for constructing the above-mentioned glucosyltransferase mutant.

[0009] The third object of the present invention is to provide a construct.

[0010] The fourth object of the present invention is to provide a mutant expression strain.

[0011] The fifth object of the present invention is to provide the use of the above-mentioned glucosyltransferase mutant and / or mutant expression strain in the production of rebaudioside M.

[0012] The sixth object of the present invention is to provide a method for preparing rebaudioside M.

[0013] Specifically, compared to the original glucosyltransferase comprising an amino acid fragment having a sequence as shown in SEQ ID NO: 1, the glucosyltransferase mutant provided by the present invention comprises one or more of L79T mutation, S195V mutation and Y387V mutation.

[0014] Furthermore, the glucosyltransferase mutant includes an L79T mutation, and the glucosyltransferase mutant includes an amino acid fragment having a sequence as shown in SEQ ID NO:2.

[0015] Furthermore, the glucosyltransferase mutant includes an S195V mutation, and the glucosyltransferase mutant includes an amino acid fragment having a sequence as shown in SEQ ID NO:3.

[0016] Furthermore, the glucosyltransferase mutant includes a Y387V mutation, and the glucosyltransferase mutant includes an amino acid fragment having a sequence as shown in SEQ ID NO:4.

[0017] Furthermore, the glucosyltransferase mutant includes L79T mutation, S195V mutation and Y387V mutation, and the glucosyltransferase mutant includes an amino acid fragment whose sequence is shown in SEQ ID NO:5.

[0018] The method for constructing a glucosyltransferase mutant provided by the present invention comprises: performing site-directed mutagenesis on the coding gene of the original glucosyltransferase to construct a construct comprising the coding gene of the glucosyltransferase mutant; transforming a host cell with the construct to obtain a mutant expression strain; and inducing expression in the mutant expression strain to obtain the glucosyltransferase mutant.

[0019] Furthermore, the original glucosyltransferase encoding gene includes a nucleotide fragment whose sequence is shown in SEQ ID NO:6.

[0020] Furthermore, the primers for site-directed mutagenesis include one or more of an L79T mutation primer pair, an S195V mutation primer pair, and a Y387V mutation primer pair.

[0021] Furthermore, the L79T mutation primer pair includes a primer L79T-F having a sequence as shown in SEQ ID NO:7 and a primer L79T-R having a sequence as shown in SEQ ID NO:8.

[0022] Furthermore, the S195V mutation primer pair includes a primer S195V-F having a sequence as shown in SEQ ID NO:9 and a primer S195V-R having a sequence as shown in SEQ ID NO:10.

[0023] Furthermore, the Y387V mutation primer pair includes a primer Y387V-F having a sequence as shown in SEQ ID NO:11 and a primer Y387V-R having a sequence as shown in SEQ ID NO:12.

[0024] The construct provided by the present invention comprises the coding gene of the above-mentioned glucosyltransferase mutant.

[0025] The mutant expression strain provided by the present invention can express the above-mentioned glucosyltransferase mutant.

[0026] The present invention also provides the use of the above-mentioned glucosyltransferase mutant and / or mutant expression strain in the production of rebaudioside M.

[0027] The preparation method of rebaudioside M provided by the present invention comprises: mixing a composite enzyme solution and a raw material solution, and performing an enzyme catalytic reaction to obtain the rebaudioside M; wherein the composite enzyme solution comprises sucrose synthase and the above-mentioned glucosyltransferase mutant, and the raw material solution comprises sucrose, rebaudioside D and uridine diphosphate.

[0028] Furthermore, the molar ratio of sucrose, rebaudioside D and uridine diphosphate in the raw material solution is (200-600):(10-100):(1-10).

[0029] Furthermore, the mass ratio of the glucosyltransferase mutant to the sucrose synthase in the complex enzyme solution is (1-10):1.

[0030] Furthermore, the sucrose synthase comprises an amino acid fragment having a sequence as shown in SEQ ID NO:13.

[0031] Furthermore, the added concentration of the composite enzyme solution is 1-5 mg / mL, and the total enzyme concentration of the composite enzyme solution is 20-60 mg / mL.

[0032] Furthermore, the temperature of the enzyme catalytic reaction is 35-60°C and the time is 24-48 hours.

[0033] Beneficial effects:

[0034] The invention is based on the original glucosyltransferase from Stevia rebaudiana (amino acid sequence as shown in SEQ ID NO: 1) for protein directed evolution, introducing L79T mutation, S195V mutation and / or Y387V mutation, optimizing the spatial structure of enzyme molecules, so as to improve its substrate binding ability and transglucosylation catalytic ability, thereby obtaining a glucosyltransferase mutant with excellent enzyme activity. The glucosyltransferase mutant can be well applied to the industrial production of rebaudioside M, and has good application prospects.

[0035] In some preferred embodiments, sucrose synthase is compounded with the above-mentioned glucosyltransferase mutant and a multi-stage enzyme catalytic reaction is carried out using sucrose, rebaudioside D and uridine diphosphate (UDP) as substrates. Sucrose synthase is used to catalyze the reaction of UDP and sucrose to produce fructose and UDP-glucose (UDPG). The generated UDP-glucose acts as a glycosyl donor for rebaudioside D and participates in the glycosylation reaction under the efficient catalysis of the glucosyltransferase mutant, thereby preparing rebaudioside M. The process of UDP and sucrose reacting to produce fructose and UDP-glucose is reversible, and the UDP-UDPG in the reaction system can be recycled. While effectively reducing the production cost, the UDP and UDP-glucose in the reaction system are in dynamic equilibrium, reducing the inhibitory effect of the substrate and product on the sucrose synthase and the glucosyltransferase mutant, which is conducive to the large-scale and low-cost production of rebaudioside M. DETAILED DESCRIPTION

[0036] The invention is based on the problem of low enzyme activity of the existing original glucosyltransferase derived from Stevia rebaudiana. After extensive and in-depth research, it is found that introducing one or more mutations of L79T mutation, S195V mutation and Y387V mutation into the original glucosyltransferase can effectively optimize the spatial structure of the enzyme molecule, improve its ability to bind to the substrate and the activity of the catalytic site, thereby obtaining a glucosyltransferase mutant with excellent enzyme activity.

[0037] In the present invention, the original glucosyltransferase specifically includes an amino acid fragment having a sequence as shown in SEQ ID NO: 1. The numbering of the amino acid residue positions in the above L79T mutation, S195V mutation and Y387V mutation is performed with reference to the amino acid sequence shown in SEQ ID NO: 1.

[0038] In the present invention, the original glucosyltransferase may also include other amino acid fragments that do not affect the glucosyl transfer catalytic activity of the amino acid fragment shown in SEQ ID NO: 1. Those skilled in the art may introduce one or more other amino acid fragments to the amino acid fragment shown in SEQ ID NO: 1 according to the contents disclosed in existing patent documents and non-patent documents to achieve other non-enzyme activity related effects such as increasing enzyme expression. That is, under the premise of not affecting the glucosyl transfer catalytic activity of the amino acid fragment shown in SEQ ID NO: 1, the introduction of other amino acid fragments is also included in the scope of the original glucosyltransferase described in the present invention.

[0039] In the present invention, the L79T mutation is defined as a mutation of the amino acid residue at position 79 of the original glucosyltransferase from leucine (L) to threonine (T).

[0040] In some specific embodiments, compared to the original glucosyltransferase, the glucosyltransferase mutant specifically includes an L79T mutation. In this case, the glucosyltransferase mutant includes an amino acid fragment whose sequence is shown in SEQ ID NO:2.

[0041] Furthermore, the glucosyltransferase mutant may also include other amino acid fragments that do not affect the glucosyl transfer catalytic activity of the amino acid fragment shown in SEQ ID NO: 2. Those skilled in the art may introduce one or more other amino acid fragments into the amino acid fragment shown in SEQ ID NO: 2 according to the contents disclosed in the existing patent literature and non-patent literature to achieve other non-enzyme activity related effects such as increasing the enzyme expression amount. That is, under the premise of not affecting the glucosyl transfer catalytic activity of the amino acid fragment shown in SEQ ID NO: 2, the introduction of other amino acid fragments is also included in the scope of the glucosyltransferase mutant described in the present invention.

[0042] In the present invention, the S195V mutation is defined as a directed mutation in which the amino acid residue at position 195 of the original glucosyltransferase is mutated from serine (S) to valine (V).

[0043] In some specific embodiments, compared to the original glucosyltransferase, the glucosyltransferase mutant specifically includes an S195V mutation. In this case, the glucosyltransferase mutant includes an amino acid fragment whose sequence is shown in SEQ ID NO:3.

[0044] Furthermore, the glucosyltransferase mutant may also include other amino acid fragments that do not affect the glucosyl transfer catalytic activity of the amino acid fragment shown in SEQ ID NO: 3. Those skilled in the art may introduce one or more other amino acid fragments into the amino acid fragment shown in SEQ ID NO: 3 according to the contents disclosed in the existing patent literature and non-patent literature to achieve other non-enzyme activity related effects such as increasing the enzyme expression amount. That is, under the premise of not affecting the glucosyl transfer catalytic activity of the amino acid fragment shown in SEQ ID NO: 3, the introduction of other amino acid fragments is also included in the scope of the glucosyltransferase mutant described in the present invention.

[0045] In the present invention, the Y387V mutation is defined as a directed mutation in which the amino acid residue at position 387 of the original glucosyltransferase is mutated from tyrosine (Y) to valine (V).

[0046] In some specific embodiments, compared to the original glucosyltransferase, the glucosyltransferase mutant specifically includes a Y387V mutation, and the glucosyltransferase mutant includes an amino acid fragment whose sequence is shown in SEQ ID NO:4.

[0047] Furthermore, the glucosyltransferase mutant may also include other amino acid fragments that do not affect the glucosyl transfer catalytic activity of the amino acid fragment shown in SEQ ID NO: 4. Those skilled in the art may introduce one or more other amino acid fragments into the amino acid fragment shown in SEQ ID NO: 4 according to the contents disclosed in the existing patent documents and non-patent documents to achieve other non-enzyme activity related effects such as increasing the enzyme expression amount. That is, under the premise of not affecting the glucosyl transfer catalytic activity of the amino acid fragment shown in SEQ ID NO: 4, the introduction of other amino acid fragments is also included in the scope of the glucosyltransferase mutant described in the present invention.

[0048] In the present invention, compared with the original glucosyltransferase, the glucosyltransferase mutant preferably includes L79T mutation, S195V mutation and Y387V mutation at the same time, and the glucosyltransferase mutant includes an amino acid fragment as shown in SEQ ID NO: 5. At this time, the simultaneous introduction of L79T mutation, S195V mutation and Y387V mutation can obtain a glucosyltransferase mutant with significantly improved enzyme activity.

[0049] Furthermore, the glucosyltransferase mutant may also include other amino acid fragments that do not affect the glucosyl transfer catalytic activity of the amino acid fragment shown in SEQ ID NO: 5. Those skilled in the art may introduce one or more other amino acid fragments into the amino acid fragment shown in SEQ ID NO: 5 according to the contents disclosed in the existing patent documents and non-patent documents to achieve other non-enzyme activity related effects such as increasing the enzyme expression amount. That is, under the premise of not affecting the glucosyl transfer catalytic activity of the amino acid fragment shown in SEQ ID NO: 5, the introduction of other amino acid fragments is also included in the scope of the glucosyltransferase mutant described in the present invention.

[0050] Based on the goal of obtaining the above-mentioned glucosyltransferase mutant, the present invention also provides a method for constructing a glucosyltransferase mutant. The construction method specifically comprises: performing site-directed mutagenesis on the coding gene of the original glucosyltransferase to construct a construct; transforming a host cell with the construct to obtain a mutant expression strain; and inducing expression of the mutant expression strain to obtain the glucosyltransferase mutant.

[0051] In the present invention, the coding gene of the original glucosyltransferase can be deduced based on its amino acid sequence, and the codon table based on which the deduction is based is well known to those skilled in the art, and is limited to being able to translate the amino acid sequence shown in SEQ ID NO: 1. The present invention does not impose any particular limitation on its specific nucleotide sequence.

[0052] In some specific embodiments, the coding gene of the original glucosyltransferase is preferably codon-optimized for E. coli; the coding gene of the original glucosyltransferase includes a nucleotide fragment with an amino acid sequence such as SEQ ID NO: 6. In this case, the expression efficiency of the original glucosyltransferase in E. coli, the most commonly used engineering bacterium in the biological field, can be effectively improved, thereby preparing a large amount of the original glucosyltransferase. In addition, the construct obtained by site-directed mutagenesis of the coding gene of the original glucosyltransferase also has a better expression effect in E. coli.

[0053] In the present invention, the site-directed mutagenesis refers to a technique of generating a predetermined mutation in the original glucosyltransferase coding gene using a mutation primer containing a predetermined mutation site.

[0054] In the present invention, the primers used in the site-directed mutagenesis specifically include one or more of an L79T mutation primer pair, an S195V mutation primer pair, and a Y387V mutation primer pair.

[0055] In the present invention, the L79T mutant primer pair is defined as a nucleotide fragment that can direct the mutation of the codon encoding the 79th leucine in the original glucosyltransferase encoding gene to a codon encoding threonine. A person skilled in the art can design the L79T mutant primer pair based on the nucleotide sequence of the original glucosyltransferase encoding gene and the threonine codon, and the present invention does not impose any particular limitation on it.

[0056] In some specific embodiments, the L79T mutation primer pair preferably includes primer L79T-F with a sequence as described in SEQ ID NO:7 and primer L79T-R with a sequence as described in SEQ ID NO:8.

[0057] In the present invention, the S195V mutant primer pair is defined as a nucleotide fragment that can direct the mutation of the codon encoding the 195th serine in the original glucosyltransferase encoding gene to a codon encoding valine. Those skilled in the art can design the S195V mutant primer pair based on the nucleotide sequence of the original glucosyltransferase encoding gene and the codon of valine, and the present invention does not impose any particular limitation on it.

[0058] In some specific embodiments, the S195V mutation primer pair preferably includes primer S195V-F with a sequence as described in SEQ ID NO:9 and primer S195V-R with a sequence as described in SEQ ID NO:10.

[0059] In the present invention, the Y387V mutant primer pair is defined as a nucleotide fragment that can direct the mutation of the codon encoding the 387th tyrosine in the original glucosyltransferase encoding gene to a codon encoding valine. A person skilled in the art can design the Y387V mutant primer pair based on the nucleotide sequence of the original glucosyltransferase encoding gene and the codon of valine, and the present invention does not impose any particular limitation on it.

[0060] In some specific embodiments, the Y387V mutation primer pair preferably includes primer Y387V-F with a sequence as described in SEQ ID NO:11 and primer Y387V-R with a sequence as described in SEQ ID NO:12.

[0061] In the present invention, the construct includes the coding gene of the glucosyltransferase mutant and may also include nucleotide sequences for realizing different functions, which may specifically be but are not limited to one or more regulatory sequences such as promoters, enhancers, transcription termination signals, polyadenylation sequences, replication origins, nucleic acid restriction sites, homologous recombination sites, poly (A) signals, etc. in coding and non-coding regions.

[0062] In the present invention, the host cell refers to a type of cell having a protein expression system for synthesizing the glucosyltransferase mutant, which is a technical means conventionally used in the biological field and is not particularly limited in the present invention.

[0063] In the present invention, transforming a host cell with the construct refers to a technique of introducing the obtained construct into a host cell to achieve amplification and expression of the construct in the host cell, which is a conventional technical means used in the biological field and is not particularly limited in the present invention.

[0064] In some specific embodiments, the method of transforming the construct into host cells specifically includes but is not limited to: one or more of the calcium chloride method, electroporation method and lambda phage infection method.

[0065] In the present invention, the induced expression refers to a technology that uses an additionally added inducer to regulate gene expression in the host cell so that the host cell can express a large amount of glucosyltransferase mutants. This is a technical means commonly used in the biological field and the present invention does not impose any particular limitation on it.

[0066] In some specific embodiments, specific examples of the inducing agent used in the inducing expression include but are not limited to: IPTG.

[0067] Based on the goal of obtaining the above-mentioned glucosyltransferase mutant, the present invention also provides a construct, which includes the coding gene of the above-mentioned glucosyltransferase mutant.

[0068] In the present invention, the coding gene of the glucosyltransferase mutant can be obtained by the above-mentioned method for constructing the glucosyltransferase mutant, or it can be deduced and synthesized according to its amino acid sequence, and the codon table based on which the deduction is based is well known to those skilled in the art, and is limited to being able to translate the glucosyltransferase mutant. The present invention does not impose any particular limitation on its specific nucleotide sequence.

[0069] Based on the goal of obtaining the above-mentioned glucosyltransferase mutant, the present invention also provides a mutant expression strain. The mutant expression strain expresses the above-mentioned glucosyltransferase mutant.

[0070] In the present invention, the mutant expression strain can be obtained by the above-mentioned method for constructing a glucosyltransferase mutant, as long as the glucosyltransferase mutant can be expressed, and the present invention does not impose any particular limitation on it.

[0071] Based on the excellent glucosyl transfer catalytic activity of the above-mentioned glucosyltransferase mutant and its application potential in the industrial large-scale production of rebaudioside M, the present invention also provides the application of the above-mentioned glucosyltransferase mutant and / or mutant expression strain in the production of rebaudioside M.

[0072] Based on the application potential of the above-mentioned glucosyltransferase mutant in the industrial large-scale production of rebaudioside M, the present invention also provides a method for preparing rebaudioside M. The preparation method comprises: mixing a composite enzyme solution and a raw material solution, and performing an enzyme catalytic reaction to obtain the rebaudioside M.

[0073] In the present invention, the composite enzyme solution includes sucrose synthase and the above-mentioned glucosyltransferase mutant. Through the cooperation of the sucrose synthase and the glucosyltransferase mutant, a multi-stage enzyme catalytic reaction is realized - sucrose synthase is used to catalyze the reaction of UDP and sucrose to generate fructose and UDP-glucose. The generated UDP-glucose serves as a glycosyl donor for rebaudioside D and participates in glycosylation under the efficient catalytic action of the glucosyltransferase mutant to obtain rebaudioside M. In addition, the cyclic changes of UDP and UDP-glucose during the reaction process can effectively solve the inhibitory effect of substrates and products on sucrose synthase and glucosyltransferase mutants while realizing the recycling of raw materials to reduce production costs, thereby achieving a more ideal enzyme catalytic effect.

[0074] In the present invention, the sucrose synthase refers to a class of enzyme molecules that have the ability to catalyze the reaction of free fructose and glucose donor UDPG to produce sucrose and its reverse reaction. Those skilled in the art can make adaptive selections from the currently known sucrose synthases, and the present invention does not impose any particular limitation on it.

[0075] In some specific embodiments, the sucrose synthase preferably includes an amino acid fragment whose sequence is shown in SEQ ID NO:13.

[0076] In some specific embodiments, the added mass ratio of the glucosyltransferase mutant and sucrose synthase in the complex enzyme solution is preferably (1-10):1, such as 1:1, 2:1, 5:1, 8:1, 10:1 or any value therebetween.

[0077] In the present invention, the raw material solution specifically includes sucrose, rebaudioside D and uridine diphosphate. Further, the added molar ratio of sucrose, rebaudioside D and uridine diphosphate is preferably (200-600):(10-100):(1-10), such as 200:13:1, 300:10:1, 600:100:1, 600:9:10 or any value therebetween.

[0078] In some specific embodiments, the raw material solution more preferably includes: 200-600 mM sucrose, such as 200 mM, 250 mM, 300 mM, 400 mM, 450 mM, 500 mM, 600 mM or any value therebetween; 10-100 mM rebaudioside D, such as 10 mM, 13 mM, 18 mM, 20 mM, 25 mM, 30 mM, 50 mM, 80 mM, 99 mM, 100 mM or any value therebetween; 1-10 mM uridine diphosphate, such as 1, 1.3, 1.8, 2, 4, 6, 10 or any value therebetween; 1-10 mM MgCl 2 , such as 1mM, 2mM, 4mM, 7mM, 9mM, 10mM or any value therebetween; 50-100mM phosphate buffer (pH=7-8), such as 50mM, 58mM, 60mM, 70mM, 75mM, 80mM, 85mM, 90mM, 100mM or any value therebetween.

[0079] In the present invention, the added concentration of the composite enzyme solution is preferably 1-5 mg / mL, such as 1 mg / mL, 1.3 mg / mL, 2 mg / mL, 2.5 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL or any value therebetween; and the total enzyme concentration of the composite enzyme solution is preferably 20-60 mg / mL, such as 20 mg / mL, 23 mg / mL, 25 mg / mL, 28 mg / mL, 30 mg / mL, 35 mg / mL, 40 mg / mL, 45 mg / mL, 60 mg / mL or any value therebetween.

[0080] In the present invention, the conditions for the enzyme-catalyzed reaction include a temperature of preferably 35 to 60°C, such as 35°C, 37°C, 40°C, 45°C, 50°C, 60°C or any value therebetween; and a time of preferably 24 to 48h, such as 24h, 30h, 36h, 42h, 48h or any value therebetween.

[0081] The amino acid and nucleotide sequences involved in the present invention are specifically shown in Table 1.

[0082] Table 1.

[0083]

[0084]

[0085]

[0086]

[0087]

[0088] Embodiments of the present invention are described in detail below, and the examples of the embodiments are intended to be used to explain the present invention, but should not be construed as limiting the present invention. In the embodiments, if specific techniques or conditions are not specified, the techniques or conditions described in the literature in this area or the product specifications are used. If the manufacturer is not specified for reagents or instruments used, they are all conventional products that can be obtained commercially.

[0089] Preparation Example

[0090] This preparation example is used to illustrate the preparation of original glucosyltransferase and sucrose synthase, which specifically includes:

[0091] (1) The gene fragment encoding the original glucosyltransferase (amino acid sequence as shown in SEQ ID NO: 1) in the stevia rebaudiana genome was codon optimized by Escherichia coli to synthesize the optimized gene fragment SrGT (nucleotide sequence as shown in SEQ ID NO: 6).

[0092] The gene fragment encoding sucrose synthase (amino acid sequence as shown in SEQ ID NO: 13) in the Acidithiobacillus genome was codon-optimized in Escherichia coli to synthesize the optimized gene fragment AcSus (nucleotide sequence as shown in SEQ ID NO: 14).

[0093] (2) After adding NdeⅠ and XhoⅠ restriction sites at both ends of the two optimized gene fragments obtained above, they were sent to Shanghai Sangon Biotechnology Co., Ltd. for synthesis, and digested with restriction endonucleases NdeⅠ and XhoⅠ (purchased from New England Biolabs) and recombined into vector pET28a to obtain recombinant plasmids SrGT-pET28a(+) and AcSus-pET28a(+); the recombinant plasmids SrGT-pET28a(+) and AcSus-pET28a(+) were transformed into Tran5ɑ competent cells (purchased from Quanshijin Company), placed in LB liquid culture medium, and cultured overnight at 37°C and 160 rpm with shaking, and a large amount of recombinant plasmids SrGT-pET28a(+) and AcSus-pET28a(+) were extracted.

[0094] (3) The recombinant plasmids SrGT-pET28a(+) and AcSus-pET28a(+) were transformed into competent Escherichia coli BL21(DE3) cells (purchased from Quanshijin Company) to obtain recombinant Escherichia coli SrGT-pET28a(+) and AcSus-pET28a(+) expressing wild-type.

[0095] (4) The wild-type recombinant E. coli SrGT-pET28a(+) and AcSus-pET28a(+) were inoculated into LB medium containing 50 mg / L kanamycin sulfate and cultured at 37°C and 200 rpm until the OD 600 The concentration of IPTG was 0.8, and IPTG was added at a final concentration of 0.1 mM. The culture was continued at 25°C for 16 h to obtain a fermentation culture medium.

[0096] (5) The fermentation culture medium was centrifuged at 4°C and 10,000 rpm for 10 min to collect the cell mud; the cell mud was resuspended in 15 mL of sodium phosphate buffer (50 mM, pH = 7.5), and the cells were ultrasonically disrupted in an ice bath for 10 min in the form of working 4 s and resting 4 s, and centrifuged at 4°C and 10,000 rpm for 10 min. The supernatant was collected and purified using a Ni-NTA affinity chromatography column (purchased from Sangon Biotech (Shanghai) Co., Ltd.) to obtain pure enzyme solution of original glucosyltransferase and pure enzyme solution of sucrose synthase.

[0097] Example 1

[0098] This example is used to illustrate a glucosyltransferase mutant L79T and its construction. The glucosyltransferase mutant L79T has an L79T mutation compared to the original glucosyltransferase provided in the preparation example, and its amino acid sequence is specifically shown in SEQ ID NO: 2. The construction of the glucosyltransferase mutant L79T specifically includes:

[0099] (1) The recombinant plasmid SrGT-pET28a(+) prepared in Preparation Example was subjected to PCR mutation amplification using an L79T mutation primer pair (including a primer L79T-F having a nucleotide sequence as shown in SEQ ID NO: 7 and a primer L79T-R having a nucleotide sequence as shown in SEQ ID NO: 8) to obtain an L79T mutation amplification product. The L79T mutation amplification product was digested with DpnⅠ at 37° C. for 2 h to remove the template and obtain an L79T mutation construct.

[0100] The PCR system is: 25 μL 2×TransStart FastPfu Fly PCR SuperMix, 1 μL of primer L79T-F (10 μM), 1 μL of primer L79T-R (10 μM), 1 μL of recombinant plasmid SrGT-pET28a (+) (50 ng / μL), and ddH 2 Add 3% HO to 50 μL.

[0101] The PCR program was as follows: pre-denaturation at 94°C for 3 min, 30 amplification cycles (denaturation at 94°C for 20 s, annealing at 55°C for 20 s, extension at 72°C for 2 min), and extension at 72°C for 10 min.

[0102] (2) The L79T mutant construct was transformed into Tran5ɑ competent cells, spread onto LB plates containing 50 μg / mL of kana resistance, and cultured overnight at 37°C. Part of the cells from each single colony were taken for PCR identification and sequencing to screen for single clones containing the L79T mutant construct.

[0103] The L79T mutant construct was extracted from a single clone containing the L79T mutant construct, and the L79T mutant construct was transformed into Escherichia coli BL21 (DE3) competent cells to obtain a glucosyltransferase mutant L79T expression strain.

[0104] (3) The glucosyltransferase mutant L79T expression strain was inoculated into LB medium containing 50 mg / L kanamycin sulfate and cultured overnight at 37°C and 200 rpm with shaking; 1% of the culture solution was inoculated into fresh LB medium containing 50 mg / L kanamycin sulfate and cultured at 37°C and 200 rpm with shaking until the OD 600 The concentration of IPTG was 0.8, and IPTG was added at a final concentration of 0.1 mM. The culture was continued at 25°C for 16 h to obtain a fermentation culture medium.

[0105] (4) The fermentation culture medium was centrifuged at 4°C and 10,000 rpm for 10 min to collect the cell mud; the cell mud was resuspended in 15 mL of sodium phosphate buffer (50 mM, pH = 7.5), and the cells were ultrasonically disrupted in an ice bath for 10 min in the form of working 4 s and resting 4 s, and centrifuged at 4°C and 10,000 rpm for 10 min. The supernatant was collected and purified using a Ni-NTA affinity chromatography column (purchased from Sangon Biotech (Shanghai) Co., Ltd.) to obtain a pure enzyme solution of the glucosyltransferase mutant L79T.

[0106] Example 2

[0107] This example is used to illustrate a glucosyltransferase mutant S195V and its construction. The glucosyltransferase mutant S195V has an S195V mutation compared to the original glucosyltransferase provided in the preparation example, and its amino acid sequence is specifically shown in SEQ ID NO:3.

[0108] In this example, the method provided in Example 1 was used to construct the above-mentioned glucosyltransferase mutant S195V, except that in step (1), a S195V mutant primer pair (including a primer S195V-F with a nucleotide sequence as shown in SEQ ID NO:9 and a primer S195V-R with a nucleotide sequence as shown in SEQ ID NO:10) was used to perform PCR mutation amplification on the recombinant plasmid SrGT-pET28a(+) prepared in the preparation example to obtain an S195V mutant construct, and in step (2), the S195V mutant construct was used to construct an expression strain of the glucosyltransferase mutant S195V, and other conditions were the same to obtain a pure enzyme solution of the glucosyltransferase mutant S195V.

[0109] Example 3

[0110] This example is used to illustrate a glucosyltransferase mutant Y387V and its construction. The glucosyltransferase mutant Y387V has a Y387V mutation compared to the original glucosyltransferase provided in the preparation example, and its amino acid sequence is specifically shown in SEQ ID NO:4.

[0111] In this example, the method provided in Example 1 was used to construct the above-mentioned glucosyltransferase mutant Y387V, except that in step (1), a Y387V mutant primer pair (including a primer Y387V-F having a nucleotide sequence as shown in SEQ ID NO: 11 and a primer S195V-R as shown in SEQ ID NO: 12) was used to perform PCR mutation amplification on the recombinant plasmid SrGT-pET28a(+) prepared in the preparation example to obtain a Y387V mutant construct, and in step (2), the Y387V mutant construct was used to construct a glucosyltransferase mutant Y387V expression strain, and other conditions were the same to obtain a pure enzyme solution of the glucosyltransferase mutant Y387V.

[0112] Example 4

[0113] This example is used to illustrate a glucosyltransferase mutant L79T / S195V / Y387V and its construction. The glucosyltransferase mutant L79T / S195V / Y387V has L79T mutation, S195V mutation and Y387V mutation compared to the original glucosyltransferase provided in the preparation example, and its amino acid sequence is specifically shown in SEQ ID NO:5.

[0114] In this example, the method provided in Example 1 was used to construct the above-mentioned glucosyltransferase mutant L79T / S195V / Y387V, except that in step (1), the recombinant plasmid SrGT-pET28a(+) prepared in the preparation example was amplified by PCR using the L79T mutation primer pair, the S195V mutation primer pair and the Y387V mutation primer pair to obtain the L79T / S195V / Y387V mutation construct, and in step (2), the L79T / S195V / Y387V mutation construct was used to construct the glucosyltransferase mutant L79T / S195V / Y387V expression strain, and other conditions were the same to obtain the glucosyltransferase mutant L79T / S195V / Y387V pure enzyme solution.

[0115] Example 5

[0116] This example is used to illustrate the enzymatic activity of the glucosyltransferase mutant provided in the above examples, and the original glucosyltransferase provided in the preparation example is used as a control. The test specifically includes:

[0117] (1) The OD values ​​of the pure enzyme solution of the glucosyltransferase mutant L79T, the pure enzyme solution of the glucosyltransferase mutant S195V, the pure enzyme solution of the glucosyltransferase mutant Y387V, the pure enzyme solution of the glucosyltransferase mutant L79T / S195V / Y387V, and the original pure enzyme solution of the glucosyltransferase provided in the preparation example were measured respectively. 280Adjust to 1.0 to obtain the enzyme solution to be tested.

[0118] (2) 5 μL of the enzyme solution to be tested was added to 200 μL of the enzyme activity assay reaction system, incubated at 35°C for 10 min, and then heated at 95°C for 5 min to quench the reaction to obtain the reaction solution to be tested; 3 times the volume of methanol was used to dilute the reaction solution to be tested, centrifuged at 20,000 g for 5 min, and filtered with a 0.22 μm filter to obtain a purified solution.

[0119] The enzyme activity assay reaction system includes 2 mM rebaudioside D, 5 mM UDPG and 50 mM Tris-HCl buffer (pH=8.0).

[0120] (3) performing high performance liquid chromatography analysis on the purified solution using a Waters Acquity UPLC system, and calculating the contents of rebaudioside D and rebaudioside M in the test reaction solution based on the standard curves of rebaudioside D and rebaudioside M, respectively;

[0121] The conditions for HPLC analysis included: the chromatographic column was a BEH C18 1.7 μm analytical column (2.1 mm × 50 mm), the column temperature was 40 °C, and the mobile phase was acetonitrile and NaH 2 PO 4 Buffer (11.5 mM, pH = 2.6); linear gradient elution method (0-1 min, 15% (v / v) acetonitrile isoconcentration elution; 1-8 min, 15-27% (v / v) acetonitrile for linear gradient elution), flow rate of 0.3 mL / min, injection volume of 10 μL, detection wavelength of 210 nm.

[0122] One enzyme activity unit (U) is defined as the amount of enzyme required to catalyze the production of 1 μmol of rebaudioside M per minute. The test results are shown in Table 2.

[0123] Table 2.

[0124] Group Enzyme activity (U / mL) Enzyme activity enhancement factor Primitive glucosyltransferase 50.0 1.00 Example 1 75.0 1.50 Example 2 112.5 2.25 Example 3 100.0 2.00 Example 4 400.0 8.00

[0125] From the test results shown in Table 2, it can be seen that compared with the original glucosyltransferase, the enzyme activity of the glucosyltransferase mutants provided in Examples 1 to 4 of the present invention is well improved, and when the L79T mutation, S195V mutation and Y387V mutation are simultaneously introduced into the original glucosyltransferase, the enzyme activity is significantly improved. The enzyme activity of the obtained glucosyltransferase is as high as 400.0 U / mL, which is 8 times higher than that of the original glucosyltransferase.

[0126] Example 6

[0127] This example is used to illustrate the preparation method of rebaudioside M, which specifically includes:

[0128] (1) The enzyme concentrations of the pure enzyme solution of sucrose synthase provided in the preparation example and the pure enzyme solution of the glucosyltransferase mutant L79T provided in Example 1 were adjusted to 50 mg / mL, and the two solutions were mixed at a volume ratio of 1:2 to obtain a composite enzyme solution.

[0129] (2) The complex enzyme solution was added to 200 mL of the raw material solution at a concentration of 2 mg / mL, and the enzyme catalytic reaction was carried out at 37°C and 220 r / min for 48 h. The reaction was quenched by heating at 95°C for 5 min to obtain an enzyme reaction solution.

[0130] The raw material solution includes 200mM sucrose, 10mM rebaudioside D, 1mM UDP, 3mM MgCl 2 , 50 mM potassium phosphate buffer (pH = 7.0).

[0131] (3) The enzyme reaction solution was diluted with 3 volumes of methanol, centrifuged at 20,000 g for 5 min, and filtered through a 0.22 μm filter to obtain a purified solution containing rebaudioside M.

[0132] Example 7

[0133] This example adopts the method provided in Example 6 to prepare rebaudioside M, except that in step (1), the mass ratio of sucrose synthase and glucosyltransferase mutant added to the complex enzyme solution is 1:1, and other conditions are the same, to obtain a purified solution containing rebaudioside M.

[0134] Example 8

[0135] This example adopts the method provided in Example 6 to prepare rebaudioside M, except that in step (1), the mass ratio of sucrose synthase and glucosyltransferase mutant added to the complex enzyme solution is 1:5, and other conditions are the same, to obtain a purified solution containing rebaudioside M.

[0136] Example 9

[0137] This example adopts the method provided in Example 6 to prepare rebaudioside M, except that in step (1), the mass ratio of sucrose synthase and glucosyltransferase mutant added to the complex enzyme solution is 1:10, and other conditions are the same, to obtain a purified solution containing rebaudioside M.

[0138] Example 10

[0139] This example adopts the method provided in Example 6 to prepare rebaudioside M, except that in step (1), an equal mass of the glucosyltransferase mutant provided in Example 2 is used in the composite enzyme solution instead of the glucosyltransferase mutant provided in Example 1, and other conditions are the same to obtain a purified solution containing rebaudioside M.

[0140] Embodiment 11

[0141] This example adopts the method provided in Example 6 to prepare rebaudioside M, except that in step (1), an equal mass of the glucosyltransferase mutant provided in Example 3 is used in the composite enzyme solution instead of the glucosyltransferase mutant provided in Example 1, and other conditions are the same to obtain a purified solution containing rebaudioside M.

[0142] Example 12

[0143] This example adopts the method provided in Example 6 to prepare rebaudioside M, except that in step (1), an equal mass of the glucosyltransferase mutant provided in Example 4 is used in the composite enzyme solution instead of the glucosyltransferase mutant provided in Example 1, and other conditions are the same to obtain a purified solution containing rebaudioside M.

[0144] Embodiment 13

[0145] This example uses the method provided in Example 6 to prepare rebaudioside M, except that in step (1), an equal mass of sucrose synthase (source leaf biotechnology, item number S10208) is used in the complex enzyme solution to replace the sucrose synthase provided in the preparation example, and other conditions are the same to obtain a purified solution containing rebaudioside M.

[0146] Embodiment 14

[0147] This example adopts the method provided in Example 6 to prepare rebaudioside M, except that in step (2), the concentration of sucrose in the raw material solution is 600 mM, the concentration of rebaudioside D is 50 mM, the concentration of UDP is 10 mM, and the concentration of MgCl is 10 mM. 2 The concentration of is 5 mM, the concentration of potassium phosphate buffer (pH = 7.4) is 75 mM, and other conditions are the same to obtain a purified solution containing rebaudioside M.

[0148] Comparative Example 1

[0149] This comparative example adopts the method provided in Example 6 to prepare rebaudioside M, except that in step (1), an equal mass of the original glucosyltransferase provided in the preparation example is used in the composite enzyme solution instead of the glucosyltransferase mutant provided in Example 1, and other conditions are the same to obtain a purified solution containing rebaudioside M.

[0150] Comparative Example 2

[0151] This comparative example adopts the method provided in Example 14 to prepare rebaudioside M, except that in step (1), an equal mass of the original glucosyltransferase provided in the preparation example is used in the composite enzyme solution instead of the glucosyltransferase mutant provided in Example 1, and other conditions are the same to obtain a purified solution containing rebaudioside M.

[0152] Embodiment 15

[0153] This example is used to illustrate the production efficiency of the preparation method of rebaudioside M provided above, and the test specifically includes: using a Waters Acquity UPLC system to perform high performance liquid chromatography analysis on the purified solution containing rebaudioside M provided in the above examples and comparative examples, and calculating the contents of rebaudioside D and rebaudioside M in the test reaction solution based on the standard curves of rebaudioside D and rebaudioside M, respectively, and obtaining the output and yield of rebaudioside M according to the following formula.

[0154] Yield of Rebaudioside M = content of Rebaudioside M × volume of purified solution

[0155] Yield = actual yield of rebaudioside M / theoretical yield of rebaudioside M × 100%

[0156] The conditions for HPLC analysis include: the chromatographic column is BEH C18 1.7 μm analytical column (2.1 mm × 50 mm), the column temperature is 40 °C, and the mobile phase is acetonitrile and NaH 2 PO 4 Buffer (11.5mM, pH=2.6); linear gradient elution (0-1min, 15% (v / v) acetonitrile isoconcentration elution; 1-8min, 15-27% (v / v) acetonitrile linear gradient elution), flow rate 0.3mL / min, injection volume 10μL, detection wavelength 210nm. The test results are shown in Table 3.

[0157] Table 3.

[0158]

[0159]

[0160] It can be seen from the test results shown in Table 3 that compared with the preparation of rebaudioside M using sucrose, rebaudioside D and UDP as raw materials using the original glucosyltransferase and sucrose synthase in Comparative Example 1, the glucosyltransferase mutant provided by the present invention has a better preparation effect, and the yield of rebaudioside M is above 70%. Compared with Comparative Example 2, the glucosyltransferase mutant provided by the present invention still shows a good preparation effect when the enzyme catalyzed reaction system is amplified, and the yield of rebaudioside M is above 70%, which has the potential to be applied to large-scale, low-cost production of rebaudioside M.

[0161] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention without departing from the principles and intent of the present invention.

Claims

1. A glucosyltransferase mutant, wherein the original glucosyltransferase comprises an amino acid fragment as shown in SEQ ID NO: 1, characterized in that: Compared to the original glucosyltransferase, the glucosyltransferase mutant includes one or more of L79T mutation, S195V mutation and Y387V mutation.

2. The glucosyltransferase mutant according to claim 1, characterized in that The glucosyltransferase mutant includes an L79T mutation, and the glucosyltransferase mutant includes an amino acid fragment whose sequence is shown in SEQ ID NO: 2; Optionally, the glucosyltransferase mutant comprises an S195V mutation, and the glucosyltransferase mutant comprises an amino acid fragment having a sequence as shown in SEQ ID NO: 3; Optionally, the glucosyltransferase mutant comprises a Y387V mutation, and the glucosyltransferase mutant comprises an amino acid fragment having a sequence as shown in SEQ ID NO: 4; Optionally, the glucosyltransferase mutant comprises an L79T mutation, an S195V mutation and a Y387V mutation, and the glucosyltransferase mutant comprises an amino acid fragment whose sequence is shown in SEQ ID NO:

5.

3. The method for constructing a glucosyltransferase mutant according to claim 1 or 2, characterized in that: The construction method comprises: performing site-directed mutagenesis on the coding gene of the original glucosyltransferase to construct a construct including the coding gene of the glucosyltransferase mutant; transforming a host cell with the construct to obtain a mutant expression strain; and inducing expression in the mutant expression strain to obtain the glucosyltransferase mutant.

4. The method for constructing a glucosyltransferase mutant according to claim 3, characterized in that: The original glucosyltransferase encoding gene includes a nucleotide fragment with a sequence as shown in SEQ ID NO: 6; Optionally, the primers for site-directed mutagenesis include one or more of an L79T mutation primer pair, an S195V mutation primer pair, and a Y387V mutation primer pair; Optionally, the L79T mutation primer pair includes a primer L79T-F having a sequence as shown in SEQ ID NO:7 and a primer L79T-R having a sequence as shown in SEQ ID NO:8; Optionally, the S195V mutation primer pair includes a primer S195V-F having a sequence as shown in SEQ ID NO:9 and a primer S195V-R having a sequence as shown in SEQ ID NO:10; Optionally, the Y387V mutation primer pair includes a primer Y387V-F having a sequence as shown in SEQ ID NO:11 and a primer Y387V-R having a sequence as shown in SEQ ID NO:

12.

5. A construct, characterized in that The construct comprises a gene encoding the glucosyltransferase mutant according to claim 1 or 2.

6. A mutant expression strain, characterized in that: The mutant expression strain is capable of expressing the glucosyltransferase mutant according to claim 1 or 2.

7. Use of the glucosyltransferase mutant according to claim 1 or 2 and / or the mutant expression strain according to claim 6 in the production of rebaudioside M.

8. A method for preparing rebaudioside M, characterized in that: The preparation method comprises: mixing a composite enzyme solution and a raw material solution, and performing an enzyme-catalyzed reaction to obtain the rebaudioside M; wherein the composite enzyme solution comprises sucrose synthase and the glucosyltransferase mutant according to claim 1 or 2, and the raw material solution comprises sucrose, rebaudioside D and uridine diphosphate.

9. The method for preparing rebaudioside M according to claim 8, characterized in that: The molar ratio of sucrose, rebaudioside D and uridine diphosphate in the raw material solution is (200-600):(10-100):(1-10); Optionally, the mass ratio of the glucosyltransferase mutant to the sucrose synthase in the complex enzyme solution is (1-10):1; Optionally, the added concentration of the complex enzyme solution is 1 to 5 mg / mL, and the total enzyme concentration of the complex enzyme solution is 20 to 60 mg / mL.

10. The method for preparing rebaudioside M according to claim 8, characterized in that: The temperature of the enzyme catalytic reaction is 35-60° C. and the time is 24-48 hours.

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